This invention relates to a nucleic acid that contains at least one nucleic acid sequence coding for a polypeptide, said polypeptide being capable of reducing the enzymatic activity of an invertase, the polypeptide itself, as well as transgenic plants that contain this nucleic acid sequence. The invention further relates to methods of preparing such transgenic plants with reduced storage sucrose loss.
During the storage of sugar beets (Beta vulgaris), in the period between harvest and processing, respiration or sucrose metabolism leads to a sucrose loss of roughly 0.02% per day. This loss is further accompanied by a significant diminution of quality as a consequence of the increase of reducing sugars, in particular fructose and glucose (Burba, M. (1976), xe2x80x9cRespiration and Sucrose Metabolism of Sugar Beets in Storage,xe2x80x9d Zeitschrift fxc3xcr die Zuckerindustrie 26:647-658). The first metabolic step in the breakdown of sucrose during the storage of beets is enzymatic hydrolysis by a vacuolar invertase. This enzyme is synthesized de novo in the beet tissue after injury (Milling, R. J., Leigh, R. A., and Hall, J. L. (1993), xe2x80x9cSynthesis of a Vacuolar Acid Invertase in Washed Discs of Storage Root Tissue of Red Beet (Beta vulgaris L.), J. Exp. Bot. 44:1687-1694). Because the bulk of beet sucrose is localized in the vacuoles of the cell, the (injury-)induced vacuolar invertase plays a central role in storage sucrose loss.
At present there is no satisfactory solution to the problem of storage sucrose losses (Burba, 1976). The most important practices in the prior art consist in maintaining low temperatures (below 12xc2x0 C.) and a well-defined atmospheric humidity (between 90 and 96%). All practices used up to now to reduce the storage losses are, however, unsatisfactory.
Conversion of sucrose to the hexoses glucose and fructose in storage, and thus loss of sucrose, also occurs during the xe2x80x9ccold sweeteningxe2x80x9d of potatoes. As a result of cold processing, a vacuolar invertase is induced in the potato tubers and determines the ratio of sucrose to hexoses (Zrenner, R., Schxc3xcler, K., and Sonnewald, U. (1996), xe2x80x9cSoluble Acid Invertase Determines the Hexose-to-Sucrose Ratio in Cold-Stored Potato Tubers,xe2x80x9d Planta 198:246-252). The formation of hexoses as a result of cold sweetening leads to diminutions of quality in the making of, for example, French-fried potatoes.
Tomato fruits (Lycopersicon esculentum Mill.) exhibit a high water content. This is due in part to the osmotically active endogenous sugars (sucrose and hexoses). Lowering the total sugar content by means of inhibiting the invertase-mediated hydrolysis of sucrose leads to smaller fruits with lower water content (Klann, E. M., Hall, B., and Bennett, A. B. (1996), xe2x80x9cAntisense Acid Invertase (TIV1) Gene Alters Soluble Sugar Composition and Size in Transgenic Tomato Fruit,xe2x80x9d Plant Physiology 112:1321-1330). Reducing the water content of the tomato fruits leads to a saving in energy costs for the production of fruit concentrates (e.g., ketchup). Because the reduction of vacuolar invertase activity via invertase antisense expression is incomplete because of the occurrence of a variety of isoforms, the transgenic introduction of an invertase inhibitor might result in great advantages, in particular if said invertase inhibitor has an equal inhibiting action on these various isoforms.
It is therefore an object of the invention to create a new system that causes essentially no sucrose storage-related losses in plants.
This object is achieved by virtue of the subject matters of the invention characterized in the Claims.
A first subject matter of the invention relates to a nucleic acid that contains at least one nucleic acid sequence coding for a polypeptide, which polypeptide is capable of reducing or lowering the enzymatic activity of an invertase.
The terms xe2x80x9cnucleic acidxe2x80x9d and xe2x80x9cnucleic acid sequencexe2x80x9d denote natural or semisynthetic or synthetic or modified nucleic acid molecules from deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides.
The term xe2x80x9cpolypeptidexe2x80x9d denotes naturally occurring polypeptides and recombinant polypeptides. Recombinant polypeptides denote a construct prepared by molecular-biological techniques, based on the natural DNA of the original genome or the natural DNA modified with a foreign DNA sequence, which construct can be recombined, for example with plasmids, and replicated and expressed in a suitable host system.
The expression xe2x80x9ca polypeptide capable of reducing the enzymatic activity of an invertasexe2x80x9d denotes a polypeptide that, in the process of binding to an invertase, reduces the enzymatic activity of said invertase, complete inhibition being possible if there is a sufficient quantity of the inhibitor protein. A roughly 90% inhibition of the vacuolar invertase is preferably to be achieved by means of the inhibitor expression in the transgenic plant.
In a embodiment of the invention, the invertase in a plant cell is vacuolarly localized. In another embodiment, the invertase is localized in the cell wall. In a further embodiment, the invertase is localized in the cytosol. The invertase is preferably derived from sugar beet, potato or tomato.
In a preferred embodiment of the invention, the nucleic acid comprises the nucleic acid sequences shown in FIGS. 1(a)-1(d) (SEQ ID No. 1), 3 (SEQ ID No. 2), 12 (SEQ ID No. 3) and 14(a)-(b) (SEQ ID No. 4) or segments or fragments thereof as well as nucleic acid sequences that can hybridize with the complementary sequences of the nucleic acid sequences shown in FIGS. 1(a-(d), 3, 12 or 14(a)-(b) or segments or fragments thereof.
In another embodiment, the nucleic acid according to the invention contains a further nucleic acid sequence coding for a targeting sequence. The term xe2x80x9ctargeting sequencexe2x80x9d denotes an amino acid sequence that mediates cellular targeting into a well-defined cellular compartment, for example targeting into the vacuoles.
In a preferred embodiment of the invention, the targeting sequence comprises the vacuolar targeting sequence of barley lectin having the following amino acid sequence:
SEQ ID NO: 9 LEGVFAEIAASNSTLVAE
In another embodiment, the nucleic acid according to the invention contains a further nucleic acid sequence coding for a signal peptide. The term xe2x80x9csignal peptidexe2x80x9d denotes a hydrophobic amino acid sequence that is recognized by the signal recognition particle (SRP). The SRP mediates the synthesis of the entire polypeptide on the rough endoplasmic reticulum (ER), with the consequence that the resulting polypeptide is released into the ER lumen.
In a further embodiment, the nucleic acid according to the invention contains a nucleic acid sequence coding for an ER retention sequence.
In a preferred embodiment, the signal peptide is derived from an invertase, preferably from cell-wall invertase from tobacco.
In another embodiment of the invention, the nucleic acid contains a further nucleic acid sequence that comprises a promoter suitable for expression in plants. This promoter or promoter sequence is preferably derived from the same plant as the invertase. In an especially preferred embodiment of the invention, the promoter is a promoter specific to potato or sugar beet.
In summary, the nucleic acid according to the invention can comprise the above-defined nucleic acid sequence coding the polypeptide and, if appropriate, the above-defined nucleic acid sequence coding a targeting sequence and/or the above-defined promoter, where all nucleic acid sequences coding an amino acid sequence are preferably arranged in the reading frame and can be degenerated in accordance with the genetic code.
A further subject matter of the invention is a vector that contains the above-defined nucleic acid according to the invention for the expression of the recombinant polypeptide in prokaryotic or eukaryotic host cells. The vector according to the invention can preferably contain suitable regulatory elements such as promoters, enhancers, termination sequences. The vector according to the invention can be, for example, an expression vector or a vector for the preferably stable integration of the nucleic acid according to the invention in to the genetic material of a host cell. A suitable expression system comprises, for example, the Ti plasmid or a binary plasmid system in Agrobacterium tumefaciens as vector for the stable integration of the nucleic acid according to the invention into the genetic material of a plant. Further, the nucleic acid according to the invention can, for example, also be inserted into the genetic material of a plant by means of the Ri plasmid of Agrobacterium rhizogenes, by means of direct gene transfer via polyethylene glycol, by means of electroporation, or by means of particle bombardment.
A further subject matter of the invention is a host cell that contains the nucleic acid according to the invention or the vector according to the invention. Suitable host cells are, for example, prokaryotes such as E. coli or eukaryotic host cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula polymorpha, Pichia pastoris and baculovirus-infected insect cells.
A further subject matter of the invention is the polypeptide itself that is coded by the above-defined nucleic acid sequence, where the nucleic acid sequence can be degenerated in accordance with the genetic code. The polypeptide according to the invention contains at least one amino acid sequence segment capable of reducing the enzymatic activity of an invertase. In an especially preferred embodiment, the polypeptide comprises the amino acid sequences shown in FIGS. 1(a)-1(d) (SEQ ID No. 5), 3 (SEQ ID No. 6), 12 (SEQ ID No. 7) and 14(a)-14(b) (SEQ ID No. 8) or segments or fragments thereof. The term xe2x80x9cpolypeptidexe2x80x9d further comprises, for example, iso forms from the same plant as well as homologous inhibitor sequences of other plant species, the homology at the protein level preferably being  greater than 70%.
In a embodiment of the invention, the polypeptide further contains an amino acid sequence arranged at the C-terminus of the polypeptide, which amino acid sequence comprises an above-defined targeting sequence and/or an ER retention sequence, for example xe2x80x9cKDEL,xe2x80x9d and/or an amino acid sequence arranged at the N-terminus of the polypeptide, which amino acid sequence comprises an above-defined signal peptide.
The nucleic acid sequence according to the invention, the vector according to the invention, and the polypeptide according to the invention can be prepared by means of prior art methods.
A further subject matter of the invention is a transgenic plant that contains at least the above-defined nucleic acid according to the invention.
The term xe2x80x9ctransgenic plantxe2x80x9d or xe2x80x9cplantxe2x80x9d comprises the entire plant as such as well as its parts, such as root, stem, leaf, organ-specific tissue or cells, its reproductive material, in particular seeds, and its embryos. This term further comprises starchy tubers and starchy roots, for example potato, sweet potato and cassava, and sugar plants, for example sugar cane and sugar beet, as well as tomato and maize.
In a preferred embodiment of the invention, the wild type of the transgenic plant is a sugar beet, a tomato or a potato.
A further subject matter of the invention relates to a method of preparing the transgenic plant according to the invention, wherein a plant cell is transformed by means of stable integration of the above-defined nucleic acid into the genetic material and the transformed plant cell is regenerated to the transgenic plant.
Methods of preparing transgenic plants are known in the prior art.
A further subject matter of the invention relates to the use of the above-defined nucleic acid for the preparation of a transgenic plant having reduced storage sucrose loss.
It can be stated according to the invention that the reduction in storage sucrose losses by means of the expression of the above-defined polypeptide as xe2x80x9cinvertase inhibitor proteinxe2x80x9d in transgenic plants represents, surprisingly, a highly specific, environmentally safe method for improving the quality of, for example, sugar beets or potato tubers. For sugar beet, a reduction in required production capacity is made possible by means of the boost in the efficiency of sugar recovery for a given level of productiveness. In the case of potato, the product quality of potatoes, in particular for the making of French-fried potatoes, is enhanced by means of the reduction in cold-induced hexose formation. In the case of tomato, the water content of the tomato fruit is lowered by means of the reduction of osmotically active hexoses.
By means of the combination of the nucleic acid sequence encoding the invertase inhibitor with a nucleic acid sequence encoding a suitable targeting sequence, correct vacuolar targeting of the expressed invertase inhibitors into the vacuoles can, for example, be achieved and thus the expression of the invertase inhibitor can be restricted in space. Further, the expression of the invertase inhibitor can be restricted in time by means of the use of promoters specific to, for example, beet or tuber.